Glutamine is an essential nutrient to support the survival and proliferation of cancer cells, and increased use of glutamine to fuel anabolic processes has been observed in a wide variety of tumors. However, as tumors grow, increased glutamine catabolism often depletes the local supply, leading tumor cells to face periods of glutamine deprivation. How tumor cells sense glutamine levels and survive these temporary periods of glutamine deprivation is unclear, but such survival is necessary for tumors to persist. Our goal is to define the molecular pathways that regulate cell survival under low glutamine conditions and evaluate the potential of simultaneously blocking both glutamine metabolism and the adaptive survival response as a novel therapeutic approach for cancer treatment. We have recently shown that glutamine deprivation leads to induction of the protein phosphatase 2A regulatory subunit B55a, thereby triggering the formation of an active PP2A complex consisting of catalytic C and scaffolding A subunits and the specifically-induced B55a subunit. This B55a- containing PP2A complex is required for tumor cell survival upon glutamine depletion. We further found that induction of B55a activates p53, an important sensor of metabolic stress. Our preliminary data suggest that B55a activates p53 through a mechanism that involves dephosphorylation of EDD, a previously unidentified substrate of B55a that is a known negative regulator of p53. In addition, we have preliminary data indicating that IKK?, the master regulator of NF-kB transcription factors, is phosphorylated upon glutamine deprivation and required for B55a induction. We therefore hypothesize that B55a is induced by the IKK? pathway to dephosphorylate EDD, thereby leading to p53 activation and cancer cell survival under low glutamine conditions. Thus, targeting both glutamine metabolism and the IKK? B55a-EDD-p53 survival pathway could be an effective therapeutic approach to kill tumor cells. To test this hypothesis, we propose three specific aims: 1) Determine the role of IKK? in B55a-mediated activation of p53 and cell survival upon glutamine deprivation; 2) Examine if B55a activates p53 upon glutamine deprivation by dephosphorylating EDD; 3) Determine the combined effect of IKK? and glutamine metabolism inhibition on growth of tumors with different p53 status. The results of the proposed studies will define the molecular mechanisms and functional impact of the IKK? B55a- p53 pathway in the metabolic adaptive response to glutamine deprivation. A wide variety of human cancer cell lines are sensitive to glutamine starvation, and reagents that impair cells' ability to use glutamine are currently being studied as novel cancer therapies. The proposed studies will provide deeper understanding of the survival pathway used by cancer cells when glutamine metabolism is blocked, and thus will reveal novel therapeutic directions for targeting both glutamine metabolism and the survival pathway in order to efficiently kill tumor cells.